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Introduction
Reproduction (photocopying) of editorial content of this journal is prohibited without publisher’s permission. 589
Journal of Chromatographic Science, Vol. 48, August 2010
were developed for quantification of piracetam from medicine The objective of this research was to develop and validate a
mixtures (7–15). Capillary electrophoretic methods are also simple, reliable, rapid, economical, and sensitive method for
reported, which offer very similar results (migration times and quantitative determination of piracetam and its related sub-
limits of quantification) to that of HPLC ones (retention times stances, degraded products, and impurities in pharmaceutical
and lower limits of quantification) (16,17). HPLC methods were formulation and in raw form. The method can be used for quan-
developed for determination of piracetam in plasma using dif- tification of piracetam with its impurities in pharmaceutical for-
ferent parameters and liquid chromatography devices. Starting mulation and to validate the procedures in order to demonstrate
with a simple determination of piracetam, after protein precipi- its good analytical and statistical parameters with respect to the
tation, a quantification limit of 1 µg/mL was obtained by using a pharmacokinetic and bioequivalence studies regulations
mobile phase of perchloric acid in a short SpheriSorb S5 amino (19–21).
column and detection at 200 nm (18). Other methods (8–15) The major advantage of the proposed methods is that four
applied extraction (liquid–liquid and solid-phase extraction) impurities and piracetam can be determined on a single chro-
using isocratic elution, similar UV detection but having different matographic system without changing the chromatographic
compositions of mobile phases and flow rates. The quantification system, which would be suitable for routine use in quality con-
limits were within 3–4 µg/mL yet with different retention times trol with regards to the wide range of concentration in plasma
differed significantly from 3.3 (14) to 6 min (12). samples and large number of samples needed to be analyzed in a
short time.
Table I. Linearity, Accuracy, and Precision of Piracetam and its
Impurities By the Proposed Method
Experimental
Added conc. Found conc. % Recovery % RSD*
Drug (ng/mL) (ng/mL) (mean) intra-day inter-day
Materials and reagents
Piracetam 50 48.46 96.92 2.687 3.12 All reagents were of analytical reagent-grade. Potassium dihy-
100 98.16 98.16 0.775 1.29 drogen orthophosphate, phosphoric acid triethyl amine, sodium
250 243.19 97.28 0.854 1.05 sulfate, sodium octane sulfonate, and HPLC-grade acetonitrile
500 489.44 97.89 3.617 2.55 were purchased from Merck (Darmstadt, Germany). Distilled
1000 991.13 99.11 1.075 2.17 and deionized water was used throughout the study. Piracetam
5000 5000.00 100.00 2.771 1.95 and impurities were obtained from UCB (Brussels, Belgium).
10000 9990.03 99.90 2.434 3.46 Nootropil 800 mg tablets were purchased from a local market.
Impurity 1 25 24.32 97.26 2.669 3.14
50 49.22 98.43 0.770 2.10
Instrumentation
100 99.55 99.55 1.498 2.60
500 493.23 98.65 0.936 1.70
The development and validation of the assay was performed on
1000 996.73 99.67 1.943 1.50 an HPLC system consisting of an LC-10 AT VP Shimadzu (Kyoto,
Japan) pump and SPD-10AV VP Shimadzu UV visible detector. A
Nucleosil C18 column (25 cm × 0.46 cm, 10 µm) was used for sep-
5000 5000.00 100.00 1.855 0.94
10000 10153.82 101.54 2.095 2.50
Impurity 2 45 44.46 98.81 3.005 2.10 aration. The chromatographic and integrated data were recorded
90 89.05 98.94 1.266 0.88 using a CBM-102 communication Bus Module Shimadzu on a P-
250 249.42 99.77 2.157 3.10 IV PC loaded with Class–GC software.
500 496.78 99.36 2.251 2.30
1000 1003.11 100.31 1.272 1.40 Chromatography
5000 5000.00 100.00 2.414 2.20
The mobile phase consisted of an aqueous solution containing
10000 10103.38 101.03 1.356 2.60
Impurity 3 34 32.97 96.96 1.632 2.20
acetonitrile–0.2 g/L solution of triethyl amine (15:85) with pH
80 78.29 97.86 2.349 2.90
6.5 adjusted with phosphoric acid. The flow rate was 1 mL/min.
250 248.68 99.47 0.856 1.50 The detections were made at 205 nm.
500 492.83 98.57 1.478 2.10
1000 993.58 99.36 1.276 2.18 Standard solutions preparation
5000 5000.00 100.00 1.034 1.24 Accurately weighed 10 mg of piracetam and its impurities
10000 10009.24 100.09 0.949 1.62 were individually transferred to 100-mL volumetric flasks sepa-
Impurity 4 55 53.20 96.73 2.841 2.05 rately, and appropriate dilution was made with the mobile phase.
100 97.22 97.22 2.332 3.14 The resulting solutions were sonicated for 25 min and filtered
250 250.71 100.28 0.864 1.58 through membrane filter. Final concentrations were
500 498.88 99.78 1.117 1.25
100.0 µg/mL. Aliquots of each solution were accordingly diluted
1000 995.04 99.50 1.411 0.94
5000 5000.00 100.00 1.096 2.28
with same solvent in order to obtain solutions with final concen-
10000 10046.00 100.46 0.799 1.66
trations of all drugs. All solutions were prepared fresh each day.
* RSD = Relative standard deviation, where n =5 for both intra- and inter-day. Calibration curves
Seven different concentration levels (50, 100, 250, 500, 1000,
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Journal of Chromatographic Science, Vol. 48, August 2010
5000, and 10,000 ng/mL) for piracetam; (25, 50, 100, 500, 1000, standard curves were prepared over a concentration range of 50,
5000, and 10,000 ng/mL) for 2-oxopyrrolidin-1-yl)acetic acid 100, 250, 500, 1000, 5000, and 10,000 ng/mL for piracetam; 25,
(impurity 1); (45, 90, 250, 500, 1000, 5000, and 10,000 ng/mL) 50, 100, 500, 1000, 5000, and 10,000 ng/mL for impurity 1; 45,
for pyrrolidin-2-one (impurity 2); (34, 80, 250, 500, 1000, 5000, 90, 250, 500, 1000, 5000, and 10,000 ng/mL for impurity 2; 34,
and 10,000 ng/mL) for methyl (2-oxopyrrolidin-1-yl)acetate 80, 250, 500, 1000, 5000, and 10,000 ng/mL for impurity 3; and
(impurity 3); and (55, 100, 250, 500, 1000, 5000, and 10,000 55, 100, 250, 500, 1000, 5000, and 10,000 ng/mL for impurity 4
ng/mL) for ethyl (2-oxopyrrolidin-1-yl)acetate (impurity 4) were (Table I). The data of peak area versus drug concentration was
obtained for each standard solution. Each solution was injected treated by linear least square regression analysis. The standard
in the chromatographic system (n = 5), and mean values of peak curves were evaluated for intra-day and inter-day reproducibility.
areas were plotted against concentrations. The curves were Linear regression characteristics of proposed method are given
adjusted by linear regression with the least mean squares in Table II.
method (22,23).
Precision and accuracy
Method validation Nootropil tablet solution mixtures containing known
The method was validated by using the ICH guideline (24). amounts of piracetam and its impurities were prepared and ana-
The selectivity, limits of detection and quantification, linearity, lyzed by HPLC. The accuracy of the method was checked for
precision, and accuracy were determined. Determination was three different concentrations. It was found that these additions
carried out using tablet formulation. The reversed-phase (RP)- were accurately reflected in their peak areas. All estimations
HPLC method that was presented has been proved successfully were repeated thrice, and the amounts of recovered drug were
in separation and simultaneous determination of all five com- calculated (Table III). The precision of the method was deter-
pounds. The developed method was validated for linearity, accu- mined as relative standard deviation (RSD) on replicate injec-
racy, precision, recovery, limit of detection, limit of tions (n = 5) of a standard solution as shown in Table III.
quantification, and ruggedness.
Limit of detection and limit of quantification
Linearity In order to estimate the limit of detection and limit of quan-
Stock solution of piracetam and its related substances (100 tification, mobile phase was injected six times, and the noise
µg/mL) were prepared individually in mobile phase. A series of level was determined. The limit of detection was calculated to be
three times the noise value and ten times the noise, which gave
Table II. Regression Characteristics of Piracetam and Impurities limit of quantification, and was also cross-checked by formulas
By the Proposed Method given below (Table IV).
3.3σ 10σ
Piracetam Impurity 1 Impurity 2 Impurity 3 Impurity 4 LOD = and LOD =
S S
r2 0.9999 0.9999 0.9999 0.9999 0.9999 where σ is the standard deviation of the lowest standard con-
Intercept 06.45 233.73 121.09 371.83 1032.7 centration and S is the slope of the standard curve. Analysis of
Slope 40.60 44.94 29.75 47.91 83.16 impurities in pharmaceutical formulation is indicated in Table V,
and all the results were found in accordance to acceptable limits.
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Tailing (symmetry) factor (T) and number of theoretical plates silanol on the surface of silica stationary phase support particles.
(N) were calculated by formulas: T = W/(2Wa) and Acidic or neutral compounds are not affected, and some basic
N = 5.545(tR/W1/2)2, where W is the peak width at 5% height from compounds are more adversely affected than others. Triethyl
baseline, Wa the peak front edge width at the same height, and amine is commonly added to mobile phases for this purpose.
W1/2 is the peak width at half height. Triethyl amine interacts strongly with silanols and inhibits them
According to the data of robustness test study, we proposed from interacting with amines in your sample.
criteria for system suitability test (tailing factors < 2, numbers of Acetonitrile–0.2 g/L solution of triethyl amine at pH 6.5 in the
theoretical plates > 4000), and repeatability (RSD) of five repli- proportion 15:85 (v/v) was found to be a suitable solvent system.
cates must not be more than 1.5 (peak area for replicate anal- When 50:50 (v/v) was employed as the mobile phase, poor reso-
ysis). It is used to verify that the resolution and repeatability of lution was obtained between the drug and its degradation
the system are adequate for the analysis of the intended drug. product.
The effect of sodium sulfate in mobile phase was also checked.
Analysis of the pharmaceutical formulation It is observed that sodium sulfate has the most important effect
To determine the content of piracetam from the Nootropil on chromatographic behavior. This effect is negative for the five
tablet label claim (piracetam 800 mg per tablet), 20 tablets equiv- separations, which means that an increase of the amount of
alent to 10 mg of piracetam were powdered, weighed accurately, sodium sulfate will decrease the resolution of the peak pairs
and transferred to a 100-mL volumetric flask. Mobile phase was studied. The effect of sodium 1-octanesulfonate in mobile phase
used for extraction. To ensure complete extraction of the drug, it was also studied and has a positive effect on the resolution of the
was sonicated for 30 min, and the solution was made up to 100 impurities, which means that an increase of the amount of ion
mL. The resulting solution was filtered, and the supernatant was pairing agent improves resolution. Initially, the experiment was
diluted to obtain the desired concentration with the mobile carried out with ion pairing before testing both sodium-1-
phase and injected. The analysis was repeated in triplicate. A octanesulonate and triethyl amine. Later, triethyl amine was
placebo tablet was also subjected to the same extraction process selected for method validation.
as discussed earlier before injection. The possibility of excipient Figure 1 shows the molecular structures of piracetam and its
interference in the analysis was studied. structurally related impurities impurity 1, impurity 2, impurity
3, and impurity 4 studied in the present investigation. All these
Figure 2. Representative chromatogram of piracetam and its four impurities. Figure 3. Representative chromatogram of piracetam (using NOOTROPIL
Peak 1 = impurity 1, peak 2 = piracetam, peak 3 = impurity 2, peak 4 = impu- tablet) and addition its four impurities. Peak 1 = impurity 1, peak 2 = pirac-
rity 3, peak 5 = impurity 4. etam, peak 3 = impurity 2, peak 4 = impurity 3, peak 5 = impurity 4.
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Journal of Chromatographic Science, Vol. 48, August 2010
materials were subjected to separation by RP-HPLC. The sepa- trations using proposed methods. The consequent RSD
ration and resolution were found to be pH-dependent. Due care amongst these values are within acceptable limits (< 3.5%).
was given to the pH of the mobile phase while standardizing the Table II shows the statistical treated linear regression data of
HPLC conditions. pH values between 6 and 7 were found to be piracetam and all impurities.
the optimum values for good separation. Acetonitrile was used The RSD values were less than 3.5%, indicating precision of
an organic solvent modifier to improve the separation. The the method. The recovery values obtained were between
effect of concentration of acetonitrile and temperature of the 98.78–101.25% for piracetam, 99.12–101.67% for impurity 1,
column on resolution was also studied. In the optimized condi- 99.09–101.77% for impurity 2, 101.05–101.27% for impurity 3,
tion, column temperature was set 40°C, but in this condition and 100.8–102.41% for impurity 4, as shown in Table III, con-
the piracetam peak was merged with a neighboring peak, so val- firming accuracy of the proposed method. The percentage of
idation was conducted at room temperature 25°C. The chro- recovery results is also presented in Table I. The excipients pre-
matographic separation was also found to be dependent on the sent in pharmaceutical dosage forms do not interfere in the
concentration of triethyl amine aqueous solution and the best analysis. The results prove specificity of the proposed methods.
separation was found to be optimum with acetonitrile–0.2 g/L Table V shows the analysis of all impurities in dosage formula-
aqueous solution of triethyl amine (15:85, v/v). The retention tion.
time of piracetam obtained 3.6 min, and its impurity 1, 2, 3, and A linearity relationship was evaluated across the range of ana-
4 were obtained at 3, 4, 7, and 13.2 min, respectively. A typical lytical procedure. The linearity was performed over the range of
chromatogram of a synthetic mixture containing piracetam concentration 50, 100, 250, 500, 1000, 5000, and 10,000 ng/mL
and its impurities is shown in Figure 2. Two sample chro- for piracetam; 25, 50, 100, 500, 1000, 5000, and 10,000 ng/mL
matograms (using Nootoopil tablet), one with the addition of for impurity 1; 45, 90, 250, 500, 1000, 5000, and 10,000 ng/mL
impurities and the other without any impurities are presented for impurity 2; 34, 80, 250, 500, 1000, 5000, and 10,000 ng/mL
in Figure 3–4, respectively. The peaks were identified by for impurity 3; and 55, 100, 250, 500, 1000, 5000, and 10,000
injecting and comparing the retention times with those of ng/mL for impurity 4. The regression line was calculated as y =
authentic standards. Reproducible peak shapes were obtained Bx + D, where x was the drug concentration (ng/mL) and y was
under the optimum conditions. It can be clearly seen that the the response (peak area expressed as AUC). D is the intercept,
shapes of these peaks are undistorted. Therefore, the symmetry and B is slope of regression line as mention in Table II. The cal-
C18 column was preferred over the other column, and it ibration curve was obtained using the linear least squares
provided better resolution of the peaks with symmetry and regression procedure; representative linear regression data of
reproducibility. five different analytes is shown in Table II. The correlation coef-
All calibration curves showed linearity over a concentration ficient (r2) value is 0.9999.
range. The correlation coefficients (r2) obtained with linear
regression of curve were above 0.9999 for every impurity and Stability of piracetam solution
piracetam. Linearity data show the concentration interval of The stability of the piracetam solutions during time was inves-
studied piracetam in which the intensity of the detector tigated. A solution of 10,000 ng/mL of piracetam was analyzed at
response is proportional to the concentration of the analyzed intervals of 0, 24, 48, and 72 h after storing the samples at room
substance. The theoretically determined values of quantitation temperature. It was found that up to 72 h the chromatographic
limits were crossed checked by actual analysis of these concen- peak area of piracetam was not significantly changed. It can be
concluded that piracetam solution was stable at least for 72 h
after its preparations. This solution was also analyzed in the
HPLC system at 2, 4, 6, and 8 days when stored at –20°C, and no
significant change was found when compared with freshly pre-
pared samples.
Conclusion
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Journal of Chromatographic Science, Vol. 48, August 2010
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